Compensation of spatial and temporal dispersion for acousto-optic multiphoton laser-scanning microscopy.

We describe novel approaches for compensating dispersion effects that arise when acousto-optic (AO) beam deflection of ultrafast laser pluses is used for multiphoton laser-scanning microscopy (MPLSM). AO deflection supports quick positioning of a laser beam to random locations, allowing high frame-rate imaging of user-selected sites of interest, in addition to conventional raster scanning. Compared to standard line-scan approaches, this results in improved signal strength (and thus increased signal-to-noise) as well as reduced photobleaching and photodamage. However, 2-D AO scanning has not yet been applied for multiphoton microscopy, largely because ultrafast laser pulses experience significant spatial and temporal dispersion while propagating through AO materials. We describe and quantify spatial dispersion, demonstrating it to be a significant barrier to achieving maximal spatial resolution. We also address temporal dispersion, which is a well-documented effect that limits multiphoton excitation efficacy, and is particularly severe for AO devices. To address both problems, we have developed a single diffraction grating scheme that reduces spatial dispersion more than three-fold throughout the field of view, and a novel four-pass stacked-prism prechirper that fully compensates for temporal dispersion while reducing by two-fold the required physical length relative to commonly employed designs. These developments enable the construction of a 2-D acousto-optic multiphoton laser-scanning microscope system.